The development of methods using capillary electrophoresis (CE) to measure and characterize intracellular nucleotide pools and metabolites continues. Our previous work has demonstrated that a 100- to 160-fold signal enhancement can be obtained for the CE analysis of mixtures of synthetic nucleotides with sample stacking, but that a marked deterioration in signal and resolution is noted for biological samples. Sample preparation methods and analysis strategies to overcome this effect, such as using solid phase extraction and controlling ionic strength, have provided some success and a 20- to 30-fold signal enhancement for biological samples. We have also characterized nucleotide drug metabolites in cellular matrices such as cultured Molt-4 and Hela cells using CE methods. We are evaluating whole-column sample stacking for the micropreparative CE isolation of trace nucleotide metabolites for structural analysis by MALDI mass spectrometry. This will have application for the determination and measurement of the active intracellular nucleotide metabolites of the nucleoside-based reverse transciptase inhibitors that used for AIDS therapy.The National Cancer Institute (NCI) Chemical Database, which contains over half a million unique structures acquired over the past 50 years, is a valuable resource for biological testing, for drug development and for chemical information applications. Many investigators use this database to identify new lead compounds for anticancer and antiviral drug development. The validity of this approach depends on the selected compounds possessing the stated structures. We have completed and validated a targeted analytical evaluation of the structural integrity and purity of compounds in the open NCI Chemical Database. This was accomplished by randomizing all structures and conducting a clustering analysis to identify 298 clusters which represented 85.8% of the database. A compound representative of each cluster was obtained for analysis. Of these compounds, 59.3% are ranked as good, 10.4% as questionable, and 30.3% as unacceptable. Designation as good indicates the structure is consistent with spectral data and the apparent purity is 80% or better. Ranking as questionable signifies the desired structure appears to be present but that the sample contains appreciable (>20%) contaminants or decomposition products. Classification as unacceptable denotes there is no spectral evidence for the presence of the desired compound or contaminants exceed 50% of the sample. A good correlation was observed between mass spectral and NMR data for structural integrity and sample homogeneity, although neither technique is applicable to all compounds. No obvious correlation between compound ranking and molecular weight, structural complexity or NSC number (database accession order) was observed. On the basis of the results obtained in this study, the structures and purity of any compounds obtained from a publically available compound should be confirmed, especially if those compounds have biological activity.